Use of derivatives of tetrahydro-beta-carbolines as antimetastatic agents

ABSTRACT

The present invention concerns the use of beta-carboline derivatives of formula I bearing at least a free or esterified carboxylic group on the piperidine ring, for the preparation of pharmaceutical compositions having antimetastatic properties.

The present application is a national stage entry of InternationalApplication No. PCT/EP97/01582 filed on Mar. 27, 1997.

The present invention concerns the use of derivatives oftetrahydro-beta-carbolines for the preparation of pharmaceuticalcompositions having antimetastatic properties.

The metastasizing tumor cells are capable to migrate from the primarytumor toward the target organs by means of a mechanism which encompassesthe penetration through the blood vessel walls, the entrance of thetumor cells into the blood f low, followed-by the successive crossing ofthe vessel's walls to reach the target organ.

The penetration through the connective tissue of the vessels isaccomplished by the degradation of the extracellular matrix by means ofthe metalloproteinases released by the resident connective tissue cells,which are activated by the tumor cells. Such a mechanism, which isshared also by the not tumor tissues, is usually in a dynamicequilibrium with the connective tissue regeneration, while it isexpressed in an uncontrolled way in the invading cells such as the tumoror inflammatory cells and it is involved in several pathologies such asrheumatoid arthritis, osteoarthritis, septic arthritis, cornea'sulcerations, epidermic or gastric ulceration, coronary thrombosis,proteinuria (WO 95/13289).

In such processes three types of metallo-proteinases are involved:collagenases, gelatinases and stromelysins. In normal conditions theirrelease and their activity are strictly controlled by endogenicproteinases-inhibitors, such as for example α₂ -macroglobulin.

Therefore, metallo-proteinase inhibitors may be useful in the treatmentof the pathological conditions above described as well as of thepathological consequences of traumas or also as contraceptive agents,since the metallo-proteinases are involved in the ovulation process andin the successive implant of the ovule on the uterine wall. Inparticular, the inhibition of the tumor metastasis by means ofmetallo-proteinase inhibitors is described in Matrisian et al., PNASUSA, 83, 9413-7 (1986); Wilhelm et al., PNAS USA, 84, 6725-29 (1987);Werb et al., J. Cell Biol., 109, 872-89 (1989); Liotta et al., Lab.Invest., 49, 636-49 (1983).

Metallo-proteinase inhibitors are described in U.S. Pat. No. 4,511,504,U.S. Pat. No. 4,568,666, U.S. Pat. No. 4,771,037, WO 95/13289.

Beta-carboline derivatives are described to possess variouspharmacological activities, such as for example antitumor activity[Anticancer Res., 13(6A), 2301-8 (1993); J. Antibiot., 46(11), 1672-7(1993); EP 357.122], antiulcer activity [WO 92/04348 (19.03.92)],antimalarial activity [J. Nat. Prod., 54(5), 1360-7 (1991)] or aredescribed as agents that enhance the absorption of antitumor drugs (JP04275221).

None of such molecules is however described to have antimetastaticactivity.

We have surprisingly found that the tetrahydro-beta-carbolines offormula (I) are endowed with a very high activity of inhibition of themetastatic process: ##STR1## wherein: R is selected in the groupcomprising hydrogen, linear or branched (C₁ -C₅)alkyl, phenyl(optionally substituted with a (C₁ -C₅)alkoxy group), --(CH₂)_(n)--COOH, wherein n is an integer from 1 to 3;

R₁ is hydrogen or a --COOR₄ group, wherein R₄ is hydrogen or (C₁-C₅)alkyl;

R₂ is hydrogen or a --COOR₄ group as above defined;

R₃ is selected in the group comprising hydrogen, halogen (chlorine,bromine, fluorine or iodine), (C₁ -C₄)alkoxy, benzyloxy.

Object of the present invention is the use of the compounds of formula(I), as antimetastatic agents and as inhibitors of the tumor invasionprocess.

Also the enantiomers, the racemates and the diastereoisomers of thecompounds of formula (I) are encompassed in the present invention, aswell as their salts with pharmaceutically acceptable acids or bases.

Preferred examples of compounds of formula (I) are:

6-methoxy-1,2,3,4-tetrahydronorharmane;

1,2,3,4-tetrahydronorharman-3-carboxylic acid;

6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid;

1-(4-methoxyphenyl)-1,2,3,4-tetrahydronorharman-3-carboxylic acid;

1-metil-1,2,3,4-tetraidronorharman-3-carboxylic acid;

1-methyl-1,2,3,4-tetrahydronorharman-1,3-dicarboxylic acid;

1-(diethylmethyl)-1,2,3,4-tetrahydronorharman-3-carboxylic acid;

(6-bromo-1,2,3,4-tetrahydronorharman-1-yl)-3-propionic acid;

1-isobutyl-1,2,3,4-tetrahydronorharman-3-carboxylic acid;

1-phenyl-1,2,3,4-tetrahydronorharman-3-carboxylic acid;

1-propyl-1,2,3,4-tetrahydronorharman-3-carboxylic acid;

1-methyl-1-methoxycarbonyl-6-benzyloxy-1,2,3,4-tetrahydronorharmane;

1-methyl-1-methoxycarbonyl-6-methoxy-1,2,3,4-tetrahydronorharmane;

1-methyl-1-methoxycarbonyl-6-hydroxy-1,2,3,4-tetrahydronorharmane;

1-methyl-1-methoxycarbonyl-6-chloro-1,2,3,4-tetrahydronorharmane;

1-methyl-1-methoxycarbonyl-6-bromo-1,2,3,4-tetrahydronorharmane;

1-methyl-1-methoxycarbonyl-1,2,3,4-tetrahydronorharmane.

The compounds encompassed in the present invention are known compoundsand are commercially available or can be obtained by extraction fromplants or synthesized according to methods reported in literature (seefor example WO 92/04348).

The compounds of the present invention have been tested in apharmacological "in vitro" test of inhibition of MMP8 (human neutrophilcollagenase). Said test provides for the determination via fluorescenceof the inhibition of the degradation of a fluorescent substrate(DNP-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH₂, M1855 Bachem) by means of thecatalytic domain of MMP8.

Reagents:

1) DNP-substrate=DNP-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH₂ (M1855 Bachem),M.W. 977.1 g/mol, concentration 25 (μM in DMSO; 2) measurement buffer=50mM TRIS/100 mM NaCl/10 mM CaCl₂.2H₂ O, adjusted at pH 7.6 withhydrochloric acid. 3) Enzyme=catalytic domain of MMP8 (92 Kda),concentration 0.055 mg/ml in TRIS buffer. Substrate and enzyme aremaintained at 0° C. with ice bath.

Inhibition assay:

Total volume=1 ml of solution kept under stirring in a cuvette.

Control: 0.98 ml DMSO

0.01 ml of DNP-substrate

0.01 ml of enzyme

Assay: 0.98 ml DMSO

0.01 ml DNP-substrate

0.01 ml of enzyme

0.01 ml of inhibitor (10 (g/ml).

It is measured the fluorescence at 346 nm both of the control solution(without inhibitor) and of the solution containing the inhibitor. Theinhibition of the catalytic activity of MMP8 results in the decrease inthe DNP-substrate lysis, with related decrease in the fluorescence ofthe solution.

The percentage of inhibition is expressed by the following formula:

    % Inhibition=100-(rel. unit/time.sub.with inhibitor /rel. unit/time.sub.control ×100)

By repeating the experiment at different concentrations of inhibitor itis possible to determine the IC₅₀ value.

Table I shows the data of enzymatic inhibition for some representativecompounds of the invention.

                  TABLE I                                                         ______________________________________                                                                        IC.sub.50                                       compound % Inhibition (conc. μg/ml) (μ/ml)                            ______________________________________                                        6-methoxy-1,2,3,4-tetrahydro-                                                                 100 (0.1)       0.06                                            norharmane                                                                    1,2,3,4-tetrahydronorharman- 100 (0.1) 0.07                                   3-carboxylic acid                                                             6-methoxy-1,2,3,4-tetrahydro- 100 (10)  0.05                                  norharman-1-carboxylic acid                                                 ______________________________________                                    

The compounds of the present invention have also shown activity in an"in vivo" test of chemoinvasion. In the test of chemoinvasion the CostarTranswell chambers for cell culture (diameter: 6.5 mm; pore size: 8 μm)are coated with 100 μl of Type IV collagen (diluted solution 50 μg/ml,then evaporation overnight). With the same procedure the chambers arecoated with a second layer of Type IV collagen (100 μl of solution atconcentration 50 μg/ml). Before use, the chambers are rinsed twice withsterile water and incubated for about 1 hour at 37° C. in a serum-freemedium (DMEM).

The human fibrosarcoma HT1080 cells are harvested by trypsin-EDTAtreatment, washed with DMEM+10% FCS and incubated for at least 30minutes at 37° C. in the same medium. The cells are then washed withserum-free DMEM and resuspended in serum-free DMEM added with 0.1% BSA(fraction V), counted and diluted to obtain a final density of 3×10⁵cell/ml.

Preincubated inserts are aspirated to remove the serum-free medium. Thelower compartment of the chambers is filled with 600 μl of DMEM+20%FCS+1% BSA (fraction V)+compound to test. 200 μl of cell suspension(6×10⁴ cells) containing the compound to test are added to the uppercompartment and the chambers are incubated at 37° C. under humidatmosphere with CO₂. After first 24 hour incubation the media from bothlower and upper compartments are replaced by fresh suspensions and thechambers are incubated for additional 24 hours.

Incubated filters are then washed with PBS, the cells are fixed 15 minin 4% paraformaldehyde, permeabilized in methanol (10 minutes, -20° C.)and stained with May-Grunwald-Giemsa. Cells which adhere to the top ofthe filters are removed with a cotton swab, filters are detached fromthe bottom of the chambers and analyzed with microscope to determine thenumber of cells on the lower side of the filters.

In a control experiment in absence of metallo-proteinase inhibitor,HT1080 cells, which overexpress metallo-proteinases, are able to degradeType IV collagen and to migrate to the lower side of the filters. In theexperiment with the inhibitor however the activity of themetallo-proteinases is partially or totally inhibited and the number ofcells which migrate to the lower side of the filters is decreased. Theresult of the experiment is expressed by the difference between thecells counted on the lower side of the filters in the control run and inthe experiment with the inhibitor.

Table II shows the data of two representative compounds of theinvention.

                  TABLE II                                                        ______________________________________                                                          chemoinvasion                                                 compound (conc., % inhibition) IC                                                                          .sub.50                                        ______________________________________                                        6-methoxy-1,2,3,4-tetrahydro-                                                                   10.sup.-6 M, 61.75                                                                         0.24                                             norharmane                                                                    6-methoxy-1,2,3,4-tetrahydro- 10.sup.-7 M, 56.5  0.2                          norharman-1-carboxylic acid                                                 ______________________________________                                    

From what it is said above it appears that the compounds of theinvention may be used in the treatment of the conditions associated withthe activity of the matrix metallo-proteinases, such as rheumatoidarthritis, osteoarthritis, septic arthritis, ulceration of the cornea,epidermic or gastric ulcerations, coronary thrombosis, proteinuria,pathological consequences of traumas or even as contraceptive agents.

The compounds of the present invention can be administered in dosesranging from 0.01 mg to 0.4 g per kilogram of body weight daily. Apreferred dosage regimen to obtain best results is that which providesfor the use from about 1 mg to about 50 mg per kilogram of body weightdaily, employing unitary doses such as to administer in 24 hours fromabout 70 mg to about 3.5 g of the active compound to a patient havingapproximately 70 kg of body weight. Such a dosage regimen may beadjusted to achieve the best therapeutical effect. For example, dosesmay be administered taking into account the therapeutical situation ofthe patient. The active compound may be administered by oral,intravenous, intramuscular or subcutaneous route.

The pharmaceutical compositions of the present invention containtherapeutical effective amounts of at least one compound of theinvention in admixture with pharmaceutically compatible excipients.

Oral compositions will generally include an inert diluent or an ediblecarrier. They can be included in gelatin capsules or compressed intotablets. Other oral administration forms are capsules, pills, elixirs,suspensions or syrups.

The tablets, pills, capsules and similar compositions can contain thefollowing ingredients (in addition to the active compound): a bindersuch as microcrystalline cellulose, tragacanth or gelatin; an excipientsuch as starch or lactose; a disintegrating agent such as alginic acid,primogel, maize starch and the like; a lubricant such as magnesiumstearate; a fluidifier such as colloidal silicon dioxide; a sweeteningagent such, as sucrose or saccharine or a flavoring agent such as mintflavor, methyl salicylate or orange flavor. When the compositionselected is in form of capsules, it can contain in addition a liquidcarrier such as a fat oil. Other compositions can contain variousmaterial which change the physical form thereof, for example coatingagents (for tablets and pills) such as sugar or shellac. The materialused in the preparation of the compositions should be pharmaceuticallypure and non toxic at the used dosages.

For the preparation of pharmaceutical compositions for the parenteraladministration, the active ingredient can be included in solutions orsuspensions, which can comprise in addition the following components: asterile diluent such as water for injections, saline solution, oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol; antioxidants suchas ascorbic acid or sodium bisulfite; chelating agents such asethylenediaminotetracetic acid; buffers such as acetates, citrates orphosphates and agents for adjusting the tonicity of the solution, suchas sodium chloride or dextrose. The parenteral preparation can beincluded in ampoules, mono-dose syringes, glass or plastic vials.

We claim:
 1. A method for inhibiting metallo-proteinase activity,comprising administering a compound selected from the group consistingof formula (I), enantiomers, racemates, diastereoisomers, salts, andpharmaceutically acceptable acids and bases thereof, to a patient inneed of such inhibition ##STR2## wherein: R is selected from the groupconsisting of hydrogen, linear or branched (C₁ -C₅) alkyl, phenyl whichis unsubstituted or substituted with a (C₁ -C₅) alkoxy group, and--(CH₂)_(n) --COOH, wherein n is an integer from 1 to 3;R₁ is hydrogenor a --COOR₄ group, wherein R₄ is hydrogen or (C₁ -C₅) alkyl; R₂ ishydrogen or a --COOR₄ group, wherein R₄ is hydrogen or (C₁ -C₅) alkyl;R₃ is selected from the group consisting of hydrogen, halogen, (C₁ -C₄)alkoxy and benzyloxy.
 2. The method according to claim 1, wherein saidhalogen is selected from the group consisting of chlorine, bromine,fluorine and iodine.
 3. The method according to claim 1, wherein saidcompound is selected from the group consistingof6-methoxy-1,2,3,4-tetrahydronorharmane;1,2,3,4-tetrahydronorharman-3-carboxylic acid;6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid;1-(4-methoxyphenyl)-1,2,3,4-tetrahydronorharman-3-carboxylic acid;1-metil-1,2,3,4-tetraidronorharman-3-carboxylic acid;1-methyl-1,2,3,4-tetrahydronorharman-1,3-dicarboxylic acid;1-(diethylmethyl)-1,2,3,4-tetrahydronorharman-3-carboxylic acid;6-bromo-1,2,3,4-tetrahydranorharman-1-yl)-3-propionic acid;1-isobutyl-1,2,3,4-tetrahydronorharman-3-carboxylic acid;1-phenyl-1,2,3,4-tetrahydronorharman-3-carboxylic acid;1-propyl-1,2,3,4-tetrahydronorharman-3-carboxylic acid;1-methyl-1-methoxycarbonyl-6-benzyloxy-1,2,3,4-tetrahydronorharmane;1-methyl-1-methoxycarbonyl-6-methoxy-1,2,3,4-tetrahydronorharmane;1-methyl-1-methoxycarbonyl-6-hydroxy-1,2,3,4-tetrahydronorharmane;1-methyl-1-methoxycarbonyl-6-chloro-1,2,3,4-tetrahydronorharmane;1-methyl-1-methoxycarbonyl-6-bromo-1,2,3,4-tetrahydronorharmane; and1-methyl-1-methoxycarbonyl-1,2,3,4-tetrahydronorharmane.
 4. The methodaccording to claim 3, wherein said compound is selected from the groupconsisting of6-methoxy-1,2,3,4-tetrahydronorharmane; 1.2,3,4-tetrahydronorharman-3-carboxylic acid;and6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid.
 5. Themethod according to claim 1, wherein said patient is suffering from acondition selected from the group consisting of rheumatoid arthritis,osteoarthritis, septic arthritis, ulceration of the cornea, epidermic orgastric ulcerations, coronary thrombosis, proteinuria, and pathologicalconsequences of traumas.
 6. The method according to claim 1, whereinsaid inhibition of metallo-proteinase activity results in the preventionof ovulation or the prevention of the implantation of an ovule on auterine wall.
 7. A method for inhibiting tumor invasion and metastases,comprising administering a compound selected from the group consistingof formula (I), enantiomers, racemates, diastereoisomers, salts, andpharmaceutically acceptable acids and bases thereof, to a patient inneed of such inhibition ##STR3## wherein: R is selected from the groupconsisting of hydrogen, linear or branched (C₁ -C₅) alkyl, phenyl whichis unsubstituted or substituted with a (C₁ -C₅) alkoxy group, and--(CH₂)_(n) --COOH, wherein n is an integer from 1 to 3;R₁ is hydrogenor a --COOR₄ group, wherein R₄ is hydrogen or (C₁ -C₅) alkyl; R₂ ishydrogen or a --COOR₄ group, wherein R₄ is hydrogen or (C₁ -C₅) alkyl;R₃ is selected from the group consisting of hydrogen, halogen, (C₁ -C₄)alkoxy and benzyloxy.
 8. The method according to claim 7, wherein saidhalogen is selected from the group consisting of chlorine, bromine,fluorine and iodine.
 9. The method according to claim 7, wherein saidcompound is selected from the group consistingof6-methoxy-1,2,3,4-tetrahydronorharmane;1,2,3,4-tetrahydronorharman-3-carboxylic acid;6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid;1-(4-methoxyphenyl)-1,2,3,4-tetrahydronorharman-3-carboxylic acid;1-metil-1,2,3,4-tetraidronorharman-3-carboxylic acid;1-methyl-1,2,3,4-tetrahydronorharman-1,3-dicarboxylic acid;1-(diethylmethyl)-1,2,3,4-tetrahydronorharman-3-carboxylic acid;(6-bromo-1,2,3,4-tetrahydronorharman-1-yl)-3-propionic acid;1-isobutyl-1,2,3,4-tetrahydronorharman-3-carboxylic acid;1-phenyl-1,2,3,4-tetrahydronorharman-3-carboxylic acid;1-propyl-1,2,3,4-tetrahydronorharman-3-carboxylic acid;1-methyl-1-methoxycarbonyl-6-benzyloxy-1,2,3,4-tetrahydronorharmane;1-methyl-1-methoxycarbonyl-6-methoxy-1,2,3,4-tetrahydronorharmane;1-methyl-1-methoxycarbonyl-6-hydroxy-1,2,3,4-tetrahydronorharmane;1-methyl-1-methoxycarbonyl-6-chloro-1,2,3,4-tetrahydronorharmane;1-methyl-1-methoxycarbonyl-6-bromo-1,2,3,4-tetrahydronorharmane; and1-methyl-1-methoxycarbonyl-1,2,3,4-tetrahydronorharmane.
 10. The methodaccording to claim 9, wherein said compound is selected from the groupconsisting of6-methoxy-1,2,3,4-tetrahydronorharmane; 1.2,3,4-tetrahydronorharinan-3-carboxylic acid;and6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid.